The use of bacteria for removal of nitrogen from wastewaters features a combination of nitrification and denitrification processes. (Tchobanoglous, G., et al., (1991) Wastewater Engineering: Treatment, Disposal, and Reuse. Boston, Mass.: Irwin/McGraw-Hill). A disadvantage of the nitrification is that large amounts of oxygen and energy are required to convert all the ammonium (NH4+) into nitrate (NO3−). The subsequent biological reduction of nitrate to nitrogen gas (N2) requires heterotrophic bacteria that utilize a carbon source to convert NO3− into N2 gas typically under anoxic conditions (Furukawa, K., et al., (2009) Innovative treatment system for digester liquor using anammox process. Bioresour. Technol. 100:5437-5443). Given the high energy costs pertaining to nitrification and the addition of carbon source pertaining to the denitrification process, there is a need to develop a more economical treatment system for effluents containing high ammonium concentrations.
An alternative biological process to N2 production via nitrite (NO2−) reduction is via anaerobic ammonia oxidation. Anaerobic ammonia oxidation is also referred to as anammox. The anammox process was recognized in a wastewater treatment system based on N mass balance (Mulder, A., et al., (1995) Anaerobic ammonium oxidation discovered in a denitrifying fluidized bed reactor. FEMS Microbiol. Ecol. 16:177-184). In the anammox process, under anaerobic and autotrophic conditions, ammonium (NH4+) serves as the electron donor using nitrite (NO2−) as the electron acceptor resulting in production of harmless di-nitrogen (N2) gas (Strous, M., et al., (1998) The sequencing batch reactor as a powerful tool for the study of slowly growing anaerobic ammonium-oxidizing microorganisms. Appl. Micobiol. Biotechnol. 50:589-596; Jetten, M. S. M., et al., (1999) The anaerobic oxidation of ammonium. FEMS Microbiol. Rev. 22:421-437). The complete ammonia removal process, or deammonification, entails two sequential reactions: partial nitritation (NH4++1.5 O2→NO2−+H2O+2 H+) and anammox (NH4++1.32 NO2−→1.02 N2+0.26 NO3−+2 H2O). Although this anammox equation does not consider other reactants related to cell synthesis (Dongen, van L. G. J. M., et al., (2001) The SHARON-Anammox process for the treatment of ammonium rich wastewater. Water Sci. Technol. 44:154-160), it has been used to describe the basic anammox process. The partial nitritation can be accomplished with the inhibition of nitrite oxidizing bacteria through limited oxygen supply (Kuai, L. P., et al., (1998) Applied and Environmental Microbiology 64:4500-4506), the use of high process temperatures, (Dongen, van L. G. J. M., et al., (2001) The Combined Sharon/Anammox Process, STOWA report, IWA Publishing, London) or enhancing free-ammonia concentration as a result of high pH and ammonium concentrations (Anthoniesen, A. C., et al., (1976) Inhibition of nitrification by ammonia and nitrous acid. Journal WPCF 48(5):835-852).
Compared to conventional nitrification-denitrification, these combined partial nitritation and anammox reactions save more than 50% of the oxygen supply for nitrification and 100% of the external organic carbon source for denitrification (Furukawa, K., et al., (2009) Innovative treatment system for digester liquor using anammox process. Bioresour. Technol. 100:5437-5443). This leads to a significant reduction in energy needs of treatment and a decrease in operational costs. In addition, by-products of anammox do not include greenhouse gases. The isolation of anammox microorganisms adapted to animal wastewater environments is of significant importance to farming systems inasmuch as excess ammonia in modern livestock production is a global problem and the use of conventional biological N removal methods is limited by cost. Thus an economical anammox based treatment is needed to facilitate greater adoption of advanced wastewater treatment technologies by wastewater facilities.
All microorganisms found responsible for the anammox reaction have been extremely difficult to isolate and no pure cultures exist (Megonigal, J. P., et al., (2005) Anaerobic metabolism: Linkages to trace gases and aerobic processes. In Biogeochemistry Vol. 8, 317-424). One of the difficulties in isolating bacteria that perform anammox process stems from the fact that the bacteria have a slow growth rate, wherein the doubling growth rate can take approximately two weeks. Another difficulty is isolating anammox bacteria in a natural environment. Yet another difficulty is to develop a specific autotrophic enrichment media that eliminates initial growing inhibition of the particular bacterial strain while mimicking natural environment conditions.
The enrichment of anammox microorganisms has been reported in several bioreactor studies (Egli, K., et al., (2001) Enrichment and characterization of an anammox bacterium from a rotating biological contactor treating ammonium-rich leachate. Arch. Microbiol. 60:135-153; Strous, M., J et al., (1999) Missing littroph identified as a new planctomycete. Nature, 400:446-449; Toh, S. K., et al., (2002) Enrichment of autotrophic anaerobic ammonium-oxidizing consortia from various wastewaters. Microb. Ecol. 43:154-167; Nkajima, J., et al., (2008) Enrichment of anammox bacteria from marine environment for the construction of a bioremediation reactor. Appl. Microbiol. Biotechnol. 77:119-116). Common to all these studies, anammox microorganisms were identified as members of the order Planctomycetales, a major division of the Bacteria, using molecular techniques such 16 rDNA and in situ hybridization (Schmid, M. C., et al., (2005) Biomarkers for insitu detection of anaerobic ammonium-oxidizing (anammox) bacteria. Appl. Environm. Microbiol. 71:1677-1684).
It is therefore an object of the invention to isolate a bacterium strain that utilizes the anammox process. It is contemplated that the bacterium strain would be used as a biotreatment of wastewaters containing ammonia.
These and other objects of the invention will become apparent as a detailed description of the representative embodiments is disclosed infra.